Nuclear hormone receptors (NHRs) modulate transcription by binding small lipophilic ligands and have a profound impact on normal cellular function and on development. Aberrant NHR function occurs frequently in cancers. This has prompted the development of therapeutic ligands targeting NHR receptors (e.g. tamoxifen in breast cancer, ATRA in promyelocytic leukemia). New natural or synthetic ligands for nuclear receptors are likely to include useful anticancer agents. We propose an innovative, high-throughput approach to identify functional ligands of nuclear receptors. We hypothesize that an in vitro nucleosome- based biosensor can identify and distinguish ligands that activate or inhibit nuclear receptor activity. The overall goal of our work is to develop the capacity to screen large chemical libraries rapidly for functional ligands to known or to orphan nuclear receptors through the use of such a biosensor. We have developed unique tools to facilitate our pursuit of these goals. These include reagents and instrumentation that enables us to follow nucleosomal remodeling at both the single molecule and population level in real-time. This R21 application focuses on the estrogen receptor (ER) signaling pathway as a model to demonstrate the power and sensitivity of this assay. In Aim One, we construct a nucleosome in which fluorophore-tagged DNA containing an estrogen response element (ERE) is wrapped around histones so that energy is transferred from the donor fluor (Cy3) to the acceptor fluor (Cy5) (fluorescence resonance energy transfer, or FRET). In Aim Two, we measure the ability of estrogen receptor agonists to specifically remodel ERE-containing nucleosomes, manifested as a loss of FRET. Nucleosome remodeling will be studied as a function of ERE sequence, ligand (agonist versus antagonist) and tissue source of cell extracts. A cell extract-free biosensor will also be developed utilizing only recombinant proteins. In Aim Three, we will adapt the nucleosomal biosensor to a high throughput format for screening libraries and validate the system using ER agonists, antagonists and nonligands. The biosensor developed in this project should enable rapid screening of libraries of putative ligands for chosen nuclear receptors. Moreover, it will enable rapid dissection of protein or DNA components of NHR pathways. This biosensor should be more rapid and versatile than cell-based reporter gene assays and more informative than assays that measure only ligand/receptor binding. [unreadable] [unreadable] [unreadable]